Purification of adenine phosphoribosyltransferase from Brassica juncea.
Adenine phosphoribosyltransferase was purified from Brassica juncea leaves approximately 4000-fold, to homogeneity. The native enzyme is a homodimer, with a Mr of 54,000. The purification involved (NH4)2SO4 fractionation, differential ultracentrifugation, and anion-exchange, hydrophobic, dye-ligand, and affinity chromatography. The purified enzyme has a pH optimum of 9.15 and a temperature optimum of 60 degrees C. Activity of the enzyme is stimulated by Mg2+ and is inhibited by sulfhydryl reagents. At the optimum pH and 37 degrees C, the apparent Km values for adenine and 5-phosphoribosyl-1-pyrophosphate were 3.8 and 15 microM, respectively. Analysis of the purified protein by isoelectric focusing revealed the presence of two isozymes with approximate isoelectric points of 5.3 and 5.4.
Moffatt
B A
BA
Department of Biology, University of Waterloo, Ontario, Canada.
Somerville
C R
CR
D000228:Adenine Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001937:Brassica / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D013816:Thermodynamics
D015636:Magnesium Chloride; D000228:Adenine Phosphoribosyltransferase